Large scale acoustic separation device

ABSTRACT

Devices for separating a host fluid from a second fluid or particulate are disclosed. The devices include an acoustic chamber, a fluid outlet at a top end of the acoustic chamber, a concentrate outlet at a bottom end of the acoustic chamber, and an inlet on a first side end of the acoustic chamber. An ultrasonic transducer and reflector create a multi-dimensional acoustic standing wave in the acoustic chamber that traps and separates particulates (e.g. cells) from a host fluid. The host fluid is collected via the fluid outlet, and the particulates are collected via the concentrate outlet. The device is a large-scale device that is able to process liters/hour, and has a large interior volume.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/249,129, filed on Aug. 26, 2016, which claims the benefit of U.S.Provisional Patent Application Ser. No. 62/211,142, filed on Aug. 28,2015; and which claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/252,068, filed on Nov. 6, 2015, and which isalso a continuation-in-part of U.S. patent application Ser. No.14/791,115, filed Jul. 2, 2015, which claims the benefit of U.S.Provisional Patent Application Ser. No. 62/020,088, filed on Jul. 2,2014; and which claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/154,672, filed on Apr. 29, 2015. The entiredisclosures of all of the above applications are hereby fullyincorporated herein by reference.

BACKGROUND

Acoustophoresis is the separation of particles and secondary fluids froma primary or host fluid using high intensity acoustic standing waves,and without the use of membranes or physical size exclusion filters. Ithas been known that high intensity standing waves of sound can exertforces on particles in a fluid when there is a differential in bothdensity and/or compressibility, otherwise known as the acoustic contrastfactor. The pressure profile in a standing wave contains areas of localminimum pressure amplitudes at its nodes and local maxima at itsanti-nodes. Depending on the density and compressibility of theparticles, they will be trapped at the nodes or anti-nodes of thestanding wave. Generally, the higher the frequency of the standing wave,the smaller the particles that can be trapped due the pressure of thestanding wave.

The separation of materials (e.g., acoustic separation of secondaryfluids from primary fluids or particles from a primary fluid stream)that have different acoustic contrast factors (a combination of densityand the speed of sound through the material) has been demonstrated atthe MEMS (micro electrical mechanical systems) scale. At the MEMS scale,conventional acoustophoresis systems rely on using half or quarterwavelength acoustic chambers, which at frequencies of a few megahertzare typically less than a millimeter in thickness, and operate at veryslow flow rates (e.g., 4/min). Such systems are not scalable since theybenefit from extremely low Reynolds number, laminar flow operation, andrequire minimal fluid dynamic optimization.

At the macro-scale, planar acoustic standing waves have been used toaccomplish this separation process. However, a single planar wave tendsto trap the particles or secondary fluid in a manner such that they canonly be separated from the primary fluid by turning off the planarstanding wave. This does not allow for continuous operation. Also, theamount of power that is needed to generate the acoustic planar standingwave tends to heat the primary fluid through waste energy.

Conventional acoustophoresis devices have thus had limited efficacy dueto several factors including heat generation, use of planar standingwaves, limits on fluid flow, and the inability to capture differenttypes of materials. It would therefore be desirable to provide systemsand methods of generating optimized particle clusters to improve gravityseparation and collection efficiency. Improved acoustophoresis devicesusing improved fluid dynamics would also be desirable, so theacoustophoresis can be a continuous process.

BRIEF DESCRIPTION

The present disclosure relates, in various embodiments, to macro-scaleacoustophoretic devices with improved fluid dynamics that can be used toimprove the separation of particles (e.g. cells) from a particle/fluidmixture. More particularly, the devices include an acoustic chambercontaining an ultrasonic transducer and a reflector that set up amulti-dimensional acoustic standing wave.

Disclosed herein are acoustophoresis devices for separating aprimary/host fluid from a secondary fluid or particulate. For example,the particulate may be cells such as Chinese hamster ovary (CHO) cells,NS0 hybridoma cells, baby hamster kidney (BHK) cells, or human cells;lymphocytes such as T cells (e.g., regulatory T-cells (Tregs), JurkatT-cells), B cells, or NK cells; their precursors, such as peripheralblood mononuclear cells (PBMCs); algae or other plant cells, bacteria,viruses, or microcarriers.

Disclosed in various embodiments are acoustophoretic devices,comprising: an acoustic chamber that includes at least one inlet at afirst end thereof; at least one fluid outlet at a top end of theacoustophoretic device; at least one concentrate outlet at a bottom endof the acoustophoretic device; at least one ultrasonic transducercoupled to the acoustic chamber, the at least one ultrasonic transducerincluding a piezoelectric material configured to be driven by a voltagesignal to create a multi-dimensional acoustic standing wave in theacoustic chamber; and a reflector across the acoustic chamber from theat least one ultrasonic transducer; wherein the acoustic chamberincludes a plan cross-sectional area defined by a length and a width,and a side cross-sectional area defined by the width and a height,wherein the length is greater than or equal to the width, and whereinthe plan cross-sectional area is greater than the side cross-sectionalarea.

The at least one inlet may be part of a dump diffuser. The at least oneinlet may include a height that spans about 60% of a height of thepiezoelectric material. A base of the at least one inlet may be locatedalong a base of the piezoelectric material. The dump diffuser mayinclude at least one inlet flow port at an upper end of a plenum, and aflow outlet at a lower end of the plenum, the flow outlet being of ashape that provides a flow direction normal to an axial direction of themulti-dimensional acoustic standing wave generated by the at least oneultrasonic transducer.

Generally, the dump diffuser is used to make the incoming flow moreuniform by reducing non-uniformities in the acoustic chamber resultingfrom gravity forces, so that the efficiency of the acoustophoreticdevice is maximized. The at least one inlet can be configured to permitingress of fluid into the acoustic chamber at a flow rate of at least800 milliliters per minute, and the fluid collector can be configured topermit egress of fluid out of the acoustic chamber at a flow rate of atleast 25 milliliters per minute.

In some embodiments, the at least one inlet includes a first inlet atthe first end of the acoustic chamber and a second inlet at a second endof the acoustic chamber opposite the first end thereof, such that inflowof fluid into the acoustic chamber is uniform and symmetrical.

Some embodiments of the acoustophoretic device further comprise a firstangled wall below the at least one inlet and leading to the at least oneconcentrate outlet, wherein the first angled wall includes an angle fromabout 11° to about 60° relative to a first horizontal plane.

The at least one transducer may be a plurality of transducers spanningthe length of the acoustic chamber. The plurality of transducers can beserially arranged in a single row. In some embodiments, the plurality oftransducers includes a first row containing at least two transducerslocated above a second row containing at least two transducers. The atleast one concentrate outlet may include a plurality of concentrateoutlets.

The acoustic chamber may include a volume of at least 40 cubic inches.

In various embodiments of the acoustophoretic device, an angled roof, aparabolically curved roof, or a hypocycloidally curved roof leads fromthe first end and a second end of the acoustic chamber to the at leastone fluid outlet. In other embodiments, the at least one fluid outlet isconnected to a central area of the acoustic chamber.

The multi-dimensional acoustic standing wave may include an axial forcecomponent and a lateral force component which are of the same order ofmagnitude.

The ultrasonic transducer may comprise: a housing having a top end, abottom end, and an interior volume; and a crystal at the bottom end ofthe housing having an exposed exterior surface and an interior surface,the crystal being able to generate acoustic waves when driven by avoltage signal. In some embodiments, a backing layer contacts theinterior surface of the crystal, the backing layer being made of asubstantially acoustically transparent material. The substantiallyacoustically transparent material can be balsa wood, cork, or foam. Thesubstantially acoustically transparent material may have a thickness ofup to 1 inch. The substantially acoustically transparent material can bein the form of a lattice. In other embodiments, an exterior surface ofthe crystal is covered by a wear surface material with a thickness of ahalf wavelength or less, the wear surface material being a urethane,epoxy, or silicone coating. The exterior surface of the crystal may alsohave wear surface formed from a matching layer or wear plate of materialadhered to the exterior surface of the crystal. The matching layer orwear plate may be composed of aluminum oxide. In yet other embodiments,the crystal has no backing layer or wear layer, i.e. the crystal is freeof a backing layer or a wear layer.

The multi-dimensional acoustic standing wave may be a three-dimensionalstanding wave.

Also disclosed in various embodiments are acoustophoretic devices,comprising: an acoustic chamber that includes at least one inlet at afirst end thereof; at least one fluid outlet at a top end of theacoustophoretic device; at least one concentrate outlet at a bottom endof the acoustophoretic device; at least one ultrasonic transducercoupled to the acoustic chamber, the at least one ultrasonic transducerincluding a piezoelectric material configured to be driven by a voltagesignal to create a multi-dimensional acoustic standing wave in theacoustic chamber; and a reflector across the acoustic chamber from theat least one ultrasonic transducer; wherein the at least one inlet is inthe form of a dump diffuser that includes a flow outlet at a lower frontend of a plenum, a first inlet flow port at an upper side end of theplenum, and a second inlet flow port at an upper rear end of the plenum.

Flow rates through the acoustic chamber can be from about 1 milliliterper minute to about 800 milliliters per minute. The devices of thepresent disclosure may be capable of separation efficiencies of 90% andmore for cell concentrations from as low as 50,000 cells per milliliterof fluid to 80,000,000 cells per milliliter of fluid.

In particular embodiments, the multi-dimensional standing wave resultsin an acoustic radiation force having an axial force component and alateral force component that are the same order of magnitude. Inparticular embodiments, the acoustic standing wave may be amulti-dimensional acoustic standing wave (e.g., a three-dimensionalacoustic standing wave). Examples of such multi-dimensional acousticstanding waves can be found in commonly owned U.S. Pat. No. 9,228,183,the entire contents of which are hereby fully incorporated by reference.

These and other non-limiting characteristics are more particularlydescribed below.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawings, which arepresented for the purposes of illustrating the exemplary embodimentsdisclosed herein and not for the purposes of limiting the same.

FIG. 1 is an exterior perspective view of a first exemplaryacoustophoretic device according to the present disclosure. The devicehas an acoustic chamber whose horizontal cross-sectional area is greaterthan its vertical cross-sectional area.

FIG. 2 is a cross-sectional view of the acoustophoretic device of FIG.12.

FIGS. 3A-3D illustrate four exemplary embodiments of roofs that form afluid path leading from the acoustic chamber of the acoustophoreticdevice to the fluid outlet(s) at the top of the device. FIG. 3Aillustrates a roof whose flat exterior surface has a different anglefrom the flat interior surface. FIG. 3B illustrates a roof whose flatexterior surface has the same angle as the flat interior surface (i.e. aroof with constant thickness). FIG. 3C illustrates a roof with ahypocycloidally curved exterior and interior surface (i.e. the fluidpath narrows very quickly). FIG. 3D illustrates a roof that forms afluid path connecting to only a central area of the acoustic chamber.

FIGS. 4A-4D illustrate exemplary arrangements for acoustophoreticdevices having one or more concentrate outlets. In devices with multipleconcentrate outlets, the outlets are evenly spaced apart from eachother. FIG. 4A illustrates a device with a base having one concentrateoutlet. FIG. 4B illustrates a device with a base having one concentrateoutlet. FIG. 4C illustrates a device with a base having threeconcentrate outlets. FIG. 4D illustrates a device with a base havingfour concentrate outlets.

FIGS. 5A-5C illustrate exemplary embodiments of a transducer assembly ofan acoustophoretic device according to the present disclosure. FIG. 5Ashows a piezoelectric transducer assembly including a total of sixrectangular transducers arranged in two rows of three transducers. FIG.5B shows a piezoelectric transducer assembly including a total of sixsquare-shaped transducers arranged side-by-side in a single row. FIG. 5Cshows a piezoelectric transducer assembly including a total of fiverectangular transducers arranged in two rows, with the upper rowincluding two transducers and the lower row including three transducers.

FIG. 6 illustrates a simulation of particle clusters being trapped byacoustic standing waves generated by the transducers of the transducerassembly of FIG. 5C.

FIG. 7A and FIG. 7B illustrate more exemplary embodiments of apiezoelectric transducer assembly of an acoustophoretic device accordingto the present disclosure. FIG. 7A shows a piezoelectric transducerassembly including a total of three rectangular transducers arrangedside-by-side in a single row. FIG. 7B shows a piezoelectric transducerassembly including a total of eight square-shaped transducers arrangedside-by-side in a single row.

FIG. 8 is a perspective view of an exemplary dump diffuser.

FIG. 9 is a side view of the exemplary dump diffuser of FIG. 10.

FIG. 10 is a front cross-sectional view of a second exemplaryacoustophoretic device according to the present disclosure. The devicealso has an acoustic chamber whose horizontal cross-sectional area isgreater than its vertical cross-sectional area.

FIG. 11 is a front exterior perspective view of the device of FIG. 10.

FIG. 12 is a rear exterior perspective view of the device of FIG. 10.

FIG. 13 is a graph showing the percent reduction/clarification (upperlines) and phase contrast microscopy (lower lines) over time of a 1.5%yeast mixture flowed at 810 mL/minute through a 9 inch by 3 inch by 2inch (length by width by height) acoustophoretic device according to thepresent disclosure having no dump diffuser and operated at 60 volts, 80volts, and 100 volts. The lighter lines with circular points representdevice using films, while the darker lines with square points representdevices not using films.

FIG. 14 is a graph showing the percent reduction/clarification (upperlines) and phase contrast microscopy (lower lines) over time of a 3%yeast mixture flowed at 810 mL/minute through a 9 inch by 3 inch by 2inch (length by width by height) acoustophoretic device according to thepresent disclosure having no dump diffuser, five alternating tangentialflow (ATF) films, and operated at 60 volts, 80 volts, and 100 volts.

FIG. 15 is a graph showing the percent reduction/clarification over timeof a 3% yeast mixture flowed at 810 mL/minute through a 9 inch by 3 inchby 2 inch (length by width by height) acoustophoretic device accordingto the present disclosure having five alternating tangential flow (ATF)films and operated at 80 volts and 100 volts. The lighter linesrepresent devices using a dump diffuser with two roles of holesaccording to the present disclosure, while the darker lines representdevices using a half-plate dump diffuser according to the presentdisclosure.

FIG. 16 is a graph showing the percent reduction/clarification (upperlines) and phase contrast microscopy (lower lines) over time of a 3%yeast mixture flowed at 810 mL/minute through a 9 inch by 3 inch by 2inch (length by width by height) acoustophoretic device according to thepresent disclosure using a half-plate dump diffuser, five alternatingtangential flow (ATF) films, and using a transducer assembly having tworows of transducers, where the top row is switched off and the bottomrow is operated at 100 volts.

FIG. 17 is an exterior perspective view of a third exemplaryacoustophoretic device according to the present disclosure. Thisembodiment notably uses a dump diffuser in which fluids enter the dumpdiffuser plenum along two different axes rather than only one axis (asin FIG. 1).

FIG. 18 is a perspective view of a side cross-section of the device ofFIG. 17.

FIG. 19 is a side cross-sectional view of the acoustophoretic device ofFIG. 19 showing additional aspects along with FIG. 18.

FIG. 20 is a front view of the acoustophoretic device of FIG. 17 withtransparent walls to show additional features.

FIG. 21 is a magnified view of the flow chamber of the device of FIG.17.

FIG. 22 is a magnified cross-sectional view of the transducer assemblyof the acoustophoretic device of FIG. 17.

FIG. 23 is a cross-sectional diagram of a conventional ultrasonictransducer.

FIG. 24 is a cross-sectional diagram of an ultrasonic transducer of thepresent disclosure. An air gap is present within the transducer, and nobacking layer or wear plate is present.

FIG. 25 is a cross-sectional diagram of an ultrasonic transducer of thepresent disclosure. An air gap is present within the transducer, and abacking layer and wear plate are present.

FIG. 26 is a graph showing the relationship of the acoustic radiationforce, gravity/buoyancy force, and Stokes' drag force to particle size.The horizontal axis is in microns (μm) and the vertical axis is inNewtons (N).

FIG. 27 is a graph of electrical impedance amplitude versus frequencyfor a square transducer driven at different frequencies.

FIG. 28A illustrates the trapping line configurations for seven peakamplitudes of an ultrasonic transducer of the present disclosure. FIG.28B is a perspective view illustrating a separator of the presentdisclosure. The fluid flow direction and the trapping lines are shown.FIG. 28C is a view from the fluid inlet along the fluid flow direction(arrow 814) of FIG. 28B, showing the trapping nodes of the standing wavewhere particles would be captured. FIG. 28D is a view taken through thetransducers face at the trapping line configurations, along arrow 816 asshown in FIG. 28B.

DETAILED DESCRIPTION

The present disclosure may be understood more readily by reference tothe following detailed description of desired embodiments and theexamples included therein. In the following specification and the claimswhich follow, reference will be made to a number of terms which shall bedefined to have the following meanings.

Although specific terms are used in the following description for thesake of clarity, these terms are intended to refer only to theparticular structure of the embodiments selected for illustration in thedrawings, and are not intended to define or limit the scope of thedisclosure. In the drawings and the following description below, it isto be understood that like numeric designations refer to components oflike function.

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise.

The term “comprising” is used herein as requiring the presence of thenamed component and allowing the presence of other components. The term“comprising” should be construed to include the term “consisting of”,which allows the presence of only the named component, along with anyimpurities that might result from the manufacture of the namedcomponent.

Numerical values should be understood to include numerical values whichare the same when reduced to the same number of significant figures andnumerical values which differ from the stated value by less than theexperimental error of conventional measurement technique of the typedescribed in the present application to determine the value.

All ranges disclosed herein are inclusive of the recited endpoint andindependently combinable (for example, the range of “from 2 grams to 10grams” is inclusive of the endpoints, 2 grams and 10 grams, and all theintermediate values). The endpoints of the ranges and any valuesdisclosed herein are not limited to the precise range or value; they aresufficiently imprecise to include values approximating these rangesand/or values.

The modifier “about” used in connection with a quantity is inclusive ofthe stated value and has the meaning dictated by the context. When usedin the context of a range, the modifier “about” should also beconsidered as disclosing the range defined by the absolute values of thetwo endpoints. For example, the range of “from about 2 to about 10” alsodiscloses the range “from 2 to 10.” The term “about” may refer to plusor minus 10% of the indicated number. For example, “about 10%” mayindicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1.

It should be noted that many of the terms used herein are relativeterms. For example, the terms “upper” and “lower” are relative to eachother in location, i.e. an upper component is located at a higherelevation than a lower component in a given orientation, but these termscan change if the device is flipped. The terms “inlet” and “outlet” arerelative to a fluid flowing through them with respect to a givenstructure, e.g. a fluid flows through the inlet into the structure andflows through the outlet out of the structure. The terms “upstream” and“downstream” are relative to the direction in which a fluid flowsthrough various components, i.e. the flow fluids through an upstreamcomponent prior to flowing through the downstream component. It shouldbe noted that in a loop, a first component can be described as beingboth upstream of and downstream of a second component.

The terms “horizontal” and “vertical” are used to indicate directionrelative to an absolute reference, i.e. ground level. However, theseterms should not be construed to require structures to be absolutelyparallel or absolutely perpendicular to each other. For example, a firstvertical structure and a second vertical structure are not necessarilyparallel to each other. The terms “top” and “bottom” are used to referto surfaces where the top is always higher than the bottom relative toan absolute reference, i.e. the surface of the earth. The terms“upwards” and “downwards” are also relative to an absolute reference;upwards is always against the gravity of the earth.

The term “parallel” should be construed in its lay sense of two surfacesthat maintain a generally constant distance between them, and not in thestrict mathematical sense that such surfaces will never intersect whenextended to infinity.

The present application refers to “the same order of magnitude.” Twonumbers are of the same order of magnitude if the quotient of the largernumber divided by the smaller number is a value of at least 1 and lessthan 10.

The term “virus” refers to an infectious agent that can only replicateinside another living cell, and otherwise exists in the form of a virionformed from a capsid that surrounds and contains DNA or RNA, and in somecases a lipid envelope surrounding the capsid.

The term “crystal” refers to a single crystal or polycrystallinematerial that is used as a piezoelectric material.

Acoustophoresis is a low-power, no-pressure-drop, no-clog, solid-stateapproach to particle removal from fluid dispersions: i.e., it is used toachieve separations that are more typically performed with porousfilters, but it has none of the disadvantages of filters. In particular,the acoustophoretic devices of the present disclosure are suitable foruse with bioreactors and operate at the macro-scale for separations inflowing systems with high flow rates. The acoustophoretic devices aredesigned to create a high intensity multi-dimensional ultrasonicstanding wave that results in an acoustic radiation force that is largerthan the combined effects of fluid drag and buoyancy or gravity, and istherefore able to trap (i.e., hold stationary) the suspended phase (i.e.cells) to allow more time for the acoustic wave to increase particleconcentration, agglomeration and/or coalescence. This is an importantdistinction from previous approaches where particle trajectories weremerely altered by the effect of the acoustic radiation force. As aresult, in the present devices, the radiation force acts as a filterthat prevents targeted particles (e.g., biological cells) from crossingthe plane of the standing wave. The trapping capability of a standingwave may be varied as desired, for example by varying the flow rate ofthe fluid, the acoustic radiation force, and the shape of theacoustophoretic device to maximize cell retention through trapping andsettling. This technology offers a green and sustainable alternative forseparation of secondary phases with a significant reduction in cost ofenergy. Excellent particle separation efficiencies have beendemonstrated for particle sizes as small as one micron. Theacoustophoretic devices of the present disclosure have the ability tocreate ultrasonic standing wave fields that can trap particles in flowfields with flow rates greater than 1 mL/minute.

The scattering of the acoustic field off the particles results in athree dimensional acoustic radiation force, which acts as athree-dimensional trapping field. The acoustic radiation force isproportional to the particle volume (e.g. the cube of the radius) whenthe particle is small relative to the wavelength. It is proportional tofrequency and the acoustic contrast factor. It also scales with acousticenergy (e.g. the square of the acoustic pressure amplitude). Forharmonic excitation, the sinusoidal spatial variation of the force iswhat drives the particles to the stable positions within the standingwaves. When the acoustic radiation force exerted on the particles isstronger than the combined effect of fluid drag force andbuoyancy/gravitational force, the particle is trapped within theacoustic standing wave field. The action of the acoustic forces (i.e.,the lateral and axial acoustic forces) on the trapped particles resultsin formation of tightly-packed clusters through concentration,clustering, clumping, agglomeration and/or coalescence of particlesthat, when reaching a critical size, settle continuously throughenhanced gravity for particles heavier than the host fluid or rise outthrough enhanced buoyancy for particles lighter than the host fluid.Additionally, secondary inter-particle forces, such as Bjerkness forces,aid in particle agglomeration.

Most biological cell types present a higher density and lowercompressibility than the medium in which they are suspended, so that theacoustic contrast factor between the cells and the medium has a positivevalue. As a result, the axial acoustic radiation force (ARF) drives thecells towards the standing wave pressure nodes. The axial component ofthe acoustic radiation force drives the cells, with a positive contrastfactor, to the pressure nodal planes, whereas cells or other particleswith a negative contrast factor are driven to the pressure anti-nodalplanes. The radial or lateral component of the acoustic radiation forceis the force that traps the cells. The radial or lateral component ofthe ARF is larger than the combined effect of fluid drag force andgravitational force. For small cells or emulsions the drag force F_(D)can be expressed as:

${\overset{\rightarrow}{F}}_{D} = {4\;\pi\;\mu_{f}{{R_{p}\left( {{\overset{\rightarrow}{U}}_{f} - {\overset{\rightarrow}{U}}_{p}} \right)}\left\lbrack \frac{1 + {\frac{3}{2}\hat{\mu}}}{1 + \hat{\mu}} \right\rbrack}}$where U_(f) and U_(p) are the fluid and cell velocity, R_(p) is theparticle radius, μ_(f) and μ_(p) are the dynamic viscosity of the fluidand the cells, and {circumflex over (μ)}=μ_(p)/μ_(f) is the ratio ofdynamic viscosities. The buoyancy force F_(B) is expressed as:F _(B)= 4/3πR _(p) ³(ρ_(f)−ρ_(p)).

For a cell to be trapped in the multi-dimensional ultrasonic standingwave, the force balance or sum of the force vectors on the cell may beassumed to be zero, and therefore an expression for lateral acousticradiation force F_(LRF) can be found, which is given by:F _(LRF) =F _(D) +F _(B).

For a cell of known size and material property, and for a given flowrate, this equation can be used to estimate the magnitude of the lateralacoustic radiation force.

One theoretical model that is used to calculate the acoustic radiationforce is based on the formulation developed by Gor'kov. The primaryacoustic radiation force F_(A) is defined as a function of a fieldpotential U, F_(A)=−∇(U),

where the field potential U is defined as

${U = {V_{0}\left\lbrack {{\frac{\left\langle p^{2} \right\rangle}{2\;\rho_{f}c_{f}^{2}}f_{1}} - {\frac{3\;\rho_{f}\left\langle u^{2} \right\rangle}{4}f_{2}}} \right\rbrack}},$and f₁ and f₂ are the monopole and dipole contributions defined by

${f_{1} = {1 - \frac{1}{\Lambda\;\sigma^{2}}}},\mspace{14mu}{f_{2} = \frac{2\left( {\Lambda - 1} \right)}{{2\;\Lambda} + 1}},$where p is the acoustic pressure, u is the fluid particle velocity, Λ isthe ratio of cell density ρ_(p) to fluid density ρ_(f), σ is the ratioof cell sound speed c_(p) to fluid sound speed c_(f), V_(o) is thevolume of the cell, and < > indicates time averaging over the period ofthe wave.

Gork'ov's model is for a single particle in a standing wave and islimited to particle sizes that are small with respect to the wavelengthof the sound fields in the fluid and the particle. It also does not takeinto account the effect of viscosity of the fluid and the particle onthe radiation force. As a result, this model cannot be used for themacro-scale ultrasonic separators discussed herein since particleclusters can grow quite large. A more complex and complete model foracoustic radiation forces that is not limited by particle size wastherefore used. The models that were implemented are based on thetheoretical work of Yurii Ilinskii and Evgenia Zabolotskaya as describedin AIP Conference Proceedings, Vol. 1474-1, pp. 255-258 (2012). Thesemodels also include the effect of fluid and particle viscosity, andtherefore are a more accurate calculation of the acoustic radiationforce. Additional in-house models have been developed to calculateacoustic trapping forces for cylindrical shaped objects, such as the“hockey pucks” of trapped particles in the standing wave, which closelyresemble a cylinder.

The lateral force of the total acoustic radiation force (ARF) generatedby the ultrasonic transducers of the present disclosure is significantand is sufficient to overcome the fluid drag force. This lateral ARF canthus be used to retain cells within the acoustic standing wave whilefluid flows past the standing wave. Additionally, as explained above,this action of the acoustic forces (i.e., lateral and axial acousticforces) on the trapped particles results in formation of tightly packedclusters through concentration, agglomeration and/or coalescence ofparticles that settle through enhanced gravity (particles heavier thanthe host fluid) or buoyancy (particles lighter than the host fluid).Relatively large solids of one material can thus be separated fromsmaller particles of a different material, the same material, and/or thehost fluid through enhanced gravitational separation.

The multi-dimensional standing wave generates acoustic radiation forcesin both the axial direction (i.e., in the direction of the standingwave, between the transducer and the reflector, perpendicular to theflow direction) and the lateral direction (i.e., in the flow direction).As the mixture flows through the acoustic chamber, particles insuspension experience a strong axial force component in the direction ofthe standing wave. Since this acoustic force is perpendicular to theflow direction and the drag force, it quickly moves the particles topressure nodal planes or anti-nodal planes, depending on the contrastfactor of the particle. The lateral acoustic radiation force then actsto move the concentrated particles towards the center of each planarnode, resulting in agglomeration or clumping. The lateral acousticradiation force component overcomes fluid drag, which permits clumps ofparticles to continually grow and then drop out of the mixture due togravity. A drop in drag per particle as the particle cluster increasesin size and drop in acoustic radiation force per particle as theparticle cluster grows in size, may be considered together orindependently in the operation of the acoustic separator device. In atleast some examples in the present disclosure, the lateral forcecomponent and the axial force component of the multi-dimensionalacoustic standing wave are of the same order of magnitude. In thisregard, it is noted that in a multi-dimensional acoustic standing wave,the axial force may have a different value than the lateral force, e.g.be weaker or stronger, or may be equal or equivalent, but the lateralforce of a multi-dimensional acoustic standing wave is greater than thelateral force of a planar standing wave, sometimes by two orders ofmagnitude or more.

An acoustophoretic filtering device can be used in at least twodifferent ways. First, the standing waves can be used to trap expressedbiomolecules (e.g. phytochemicals, recombinant proteins or monoclonalantibodies) and separate this desired product from the cells, celldebris, and media. The expressed biomolecules can then be diverted andcollected for further processing. Second, the standing waves can be usedto trap the cells and cell debris present in the cell culture media. Thecells and cell debris, having a positive contrast factor, move to thenodes (as opposed to the anti-nodes) of the standing wave. As the cellsand cell debris agglomerate at the nodes of the standing wave, there isalso a physical scrubbing of the cell culture media that occurs wherebymore cells are trapped as they come in contact with the cells that arealready held within the standing wave. This generally separates thecells and cellular debris from the cell culture media. When the cells inthe standing wave agglomerate to the extent where the mass is no longerable to be held by the acoustic wave, the aggregated cells and cellulardebris that have been trapped can fall out of the fluid stream throughgravity, and can be collected separately. To aid this gravitationalsettling of the cells and cell debris, the standing wave may beinterrupted to allow all of the cells to fall out of the fluid streamthat is being filtered. This process can be useful for dewatering. Theexpressed biomolecules may have been removed beforehand, or remain inthe fluid stream (i.e. cell culture medium).

In the present disclosure, a perfusion bioreactor can also be used togenerate cells that can subsequently be used for various applications,including cell therapy. In this type of process, the biological cells tobe used in the cell therapy are cultured in the bioreactor and expanded(i.e. to increase the number of cells in the bioreactor through cellreproduction). These cells may be lymphocytes such as T cells (e.g.,regulatory T-cells (Tregs), Jurkat T-cells), B cells, or NK cells; theirprecursors, such as peripheral blood mononuclear cells (PBMCs); and thelike. In the perfusion bioreactor, the cell culture media (aka hostfluid), containing some cells, is sent from the bioreactor to afiltering device that produces an acoustic standing wave. A majority ofthe cells are trapped and held in the acoustic standing wave, while theremaining host fluid and other cells in the host fluid are returned tothe bioreactor. As the quantity of trapped cells increases, they formlarger clusters that will fall out of the acoustic standing wave at acritical size due to gravity forces. The clusters can fall into aconcentrate outlet outside a region of the acoustic standing wave, suchas below the acoustic standing wave, from which the cells can berecovered for use in cell therapy. Only a small portion of the cells aretrapped and removed from the bioreactor via the concentrate outlet, andthe remainder continue to reproduce in the bioreactor, allowing forcontinuous production and recovery of the desired cells.

In these applications, the acoustophoretic devices of the presentdisclosure can act as a cell retention device. The systems describedherein operate over a range of cell recirculation rates, efficientlyretain cells over a range of perfusion (or media removal) rates, and canbe tuned to fully retain or selectively pass some percentage of cellsthrough fluid flow rate, transducer power or frequency manipulation.Power and flow rates can all be monitored and used as feedback in anautomated control system.

The cells of interest may also be held in the flow chamber of theexternal filtering device through the use of an acoustic standing wavesuch that other moieties may be introduced in close proximity to and forthe purpose of changing the target cells. Such an operation wouldinclude the trapping of T cells and the subsequent introduction ofmodified lentivirus materials with a specific gene splice such that thelentivirus with a specific gene splice will transfect the T cell andgenerate a chimeric antigen receptor T cell also known as a CAR-T cell.

The acoustic filtering devices of the present disclosure are designed tomaintain a high intensity three-dimensional acoustic standing wave. Thedevice is driven by a function generator and amplifier (not shown). Thedevice performance is monitored and controlled by a computer. It may bedesirable, at times, due to acoustic streaming, to modulate thefrequency or voltage amplitude of the standing wave. This modulation maybe done by amplitude modulation and/or by frequency modulation. The dutycycle of the propagation of the standing wave may also be utilized toachieve certain results for trapping of materials. In other words, theacoustic beam may be turned on and shut off at different frequencies toachieve desired results.

The acoustophoretic devices of the present disclosure can handle higherflow rates and larger flow volumes compared to conventional devices. Afirst exemplary embodiment of an acoustophoretic device 100 forseparating a primary/host fluid from a second fluid or particulate isillustrated in FIG. 1 and FIG. 2. FIG. 1 is an exterior perspectiveview, and FIG. 2 is a front cross-sectional view of the device. Thedesign of the device provides a vertical plane or line of flow symmetry,so that a more uniform flow for the fluid through the device occurs.

Turning first to FIG. 1, the acoustophoretic device 100 is formed from asidewall 110. As illustrated here, the sidewall 110 has a rectangularshape, so that the device has a first side end 122; a second side end124 spaced apart from and opposite the first side end 122; a front side126; a rear side 128 spaced apart from and opposite the front side 126;a top end 130; and a bottom end 132 that is spaced apart from andopposite the top end 130. Also illustrated here is a support frame 160for the device. Legs 162 are shown extending from the support frame. Thesupport frame can be integral with, or a separate structure from, thedevice 100.

Referring now to FIG. 2, a roof 140 is located on top of the sidewall110, and a base 150 is located below the sidewall 110. Together, thesidewall 110, roof 140, and the base 150 enclose an interior volume 107.At least one concentrate outlet 116 is located at the bottom end of thedevice 100. As will be explained further herein, concentratedparticulates will exit the interior volume 107 through the concentrateoutlet(s). The base is illustrated as having two angled walls 152 thattaper down to the concentrate outlet 116. It is noted that these walls152 appear as straight lines due to the cross-sectional view—in threedimensions, the walls are conical.

At least one fluid inlet 112 is present at the first side end 122, whichpermits fluid to enter from the exterior of the device 100 into theinterior volume 107. As illustrated here, two fluid inlets 112 arepresent, one on each of the side ends 122, 124. At least one fluidoutlet 114 is present at the top end of the device 100. As will beexplained further herein, fluid will exit the interior volume 107through the fluid outlet(s). The fluid inlets 112 and fluid outlet 114are also visible in FIG. 1.

Referring now to FIG. 1 and FIG. 2 together, at least one ultrasonictransducer 106 and at least one reflector 108 are located on oppositesides of the interior volume, and an acoustic chamber 120 is presentbetween them. As illustrated here, three ultrasonic transducers 106 arelocated on the rear side 128 of the device, and the reflector(s) 108 arelocated on the front side 126 of the device.

It is noted that the volume of the acoustic chamber 120 and the interiorvolume 107 are not coextensive. The volume of the acoustic chamber isdefined by the sidewall 110. In contrast, the interior volume 107 alsoincludes volume from the roof 140 and the base 150. It is also notedthat the angled walls 152 have an interior angle A measured relative toa horizontal plane (defined here by the bottom 121 of the acoustic),with the angle A being in embodiments from about 10° to about 60°,including about 30° to about 45°.

Referring still to FIG. 1 and FIG. 2 together, the acoustic chamber 120has a length 101 between the first side end and the second side end; awidth 103 between the front side and the rear side; and a height 105that is defined by the height of the ultrasonic transducer(s). Thelength 101 and the width 103 thus define a plan cross-sectional area(i.e. a horizontal cross-sectional area), while the width 103 and theheight 105 define a side cross-sectional area (i.e. a verticalcross-sectional area). As seen here, the plan cross-sectional area isgreater than the side cross-sectional area.

In particular embodiments, the acoustic chamber 120 can have a volume ofat least 40 cubic inches, such that large volumes of fluid can beprocessed within the acoustic chamber. In this regard, the fluidinlet(s) 112 of the device can be configured to permit the ingress offluid into the acoustic chamber at a flow rate of at least 800milliliters per minute (mL/min).

FIGS. 3A-3D are front views of four different roofs 140 that can be usedin the acoustophoretic device. The roof forms a fluid path leading fromthe acoustic chamber 120 of the acoustophoretic device to the fluidoutlet(s) 114 at the top of the device. In these figures, each roof 140has an interior surface 142 and an exterior surface 144. It is notedthat other roof shapes and configurations can also be used, as will beseen later herein. In particular embodiments, the fluid outlets can beconfigured to permit the egress of fluid out of the acoustic chamber ata flow rate of at least 25 mL/min.

FIG. 3A illustrates a roof 140 with a flat exterior surface 144 that hasa different angle from the flat interior surface 142, such that the roofis thicker near the fluid outlet 114. The interior surface 142 extendsfrom the fluid outlet 114 to a length 141 that is about the same as thelength 101 of the acoustic chamber 107 of FIG. 2.

FIG. 3B illustrates a roof 140 whose flat exterior surface 144 has thesame angle as the flat interior surface 142, i.e. the roof has aconstant thickness. Again, the interior surface 142 extends from thefluid outlet 114 to a length 141 that is about the same as the length101 of the acoustic chamber 107 of FIG. 2.

FIG. 3C illustrates a roof 140 having a hypocycloidally curved interiorsurface 142 and exterior surface 144. Again, the interior surface 142extends from the fluid outlet 114 to a length 141 that is about the sameas the length 101 of the acoustic chamber 107 of FIG. 2. Thehypocycloidal shape of the interior surface causes the fluid path tonarrow very quickly up to the fluid outlet 114.

FIG. 3D illustrates a roof 140 with an interior surface 142 that extendsto only a short length 141. In this embodiment, the length 141 is muchshorter than the length 101 of the acoustic chamber, such that fluidonly exits to the fluid outlet 114 from a central area of the acousticchamber.

FIGS. 4A-4D are front views of four different bases 150 that can be usedin the acoustophoretic device. The base forms a fluid path leading fromthe acoustic chamber 120 of the acoustophoretic device to theconcentrate outlet(s) 116 at the bottom of the device. It is noted thatother shapes and configurations can also be used for the base, as willbe seen later herein. Legs 162 are also seen here, though again they donot need to be integral with the base.

FIG. 4A illustrates a base having one concentrate outlet 116. Two angledwalls 152 lead from the sides of the acoustic chamber to the concentrateoutlet 116.

FIG. 4B also illustrates a base having one concentrate outlet 116. Theangled walls 152 here are shallower than those in FIG. 4A.

FIG. 4C illustrates a base having three concentrate outlets 116. Theoutlets 116 are spaced evenly apart from each other. Angled walls 152lead to each concentrate outlet.

FIG. 4D illustrates a base having four concentrate outlets. The outlets116 are spaced evenly apart from each other. Angled walls 152 lead toeach concentrate outlet.

FIGS. 5A-5C show three different embodiments of a transducer assemblyformed from a plurality of ultrasonic transducers, which can be used inthe acoustophoretic devices of the present disclosure. A plurality oftransducers allows for greater particle capture efficiency, especiallywhen the transducers have different resonance frequencies, so that alarger range of particle (e.g. cell) sizes is captured. These transducerassemblies 170 are oriented along the length of the acoustic chamber,and the side ends 122, 124 and the top end 130 are labeled in eachfigure for orientation of the assembly.

FIG. 5A shows a piezoelectric transducer assembly 170 including a totalof six rectangular transducers 106 arranged in two rows 172, 174 ofthree transducers. It is contemplated that in this arrangement thetransducers collectively span the entire width and height of theacoustic chamber.

FIG. 5B shows a piezoelectric transducer assembly 170 including a totalof six square-shaped transducers 106 arranged side-by-side in a singlerow 172. The transducers collectively span the entire width of thetransducer assembly, but not the entire height of the transducerassembly.

FIG. 5C shows a piezoelectric transducer assembly 170 including a totalof five rectangular transducers 106 arranged in two rows, with the upperrow 174 including two transducers and the lower row 172 including threetransducers. It is noted that the transducers in the upper row 174 arestaggered/offset with respect to the transducers in the lower row 172.One benefit of this arrangement is illustrated in FIG. 6, whichindicates the location of multi-dimensional acoustic standing waves 176that will be generated by the transducers. As can be seen here, thestaggering of the transducers causes the acoustic standing waves 176 toalso be staggered, so that the standing waves generated by the upper row174 are staggered from the standing waves in the lower row 172. Aspreviously explained, particles/cells that are trapped in themulti-dimensional acoustic standing waves will agglomerate and formclusters that eventually fall out of the standing waves and traveldownwards towards the concentrate outlet. This staggering permits theclusters that fall downwards from the upper row 174 to avoid passingthrough the acoustic standing waves generated in the lower row 172, sothat the clusters being formed in the lower row 172 are not disturbed ordisrupted.

It is also contemplated that the plurality of transducers can bearranged serially in a single row, such as in FIG. 7A and FIG. 7B. FIG.7A shows a transducer assembly 170 including a total of threerectangular transducers 106 arranged side-by-side in a single row. FIG.7B shows a transducer assembly 170 with a total of eight square-shapedtransducers 106 arranged side-by-side in a single row.

Referring now back to FIG. 1 and FIG. 2, the device 100 has symmetricalfluid inlets 112 placed on opposite sides of the acoustic chamber. Inparticular embodiments, these inlets are in the form of dump diffusers,which provide a more uniform flow of the mixture of host fluid andparticulate into the acoustic chamber.

Briefly, each dump diffuser includes an entrance port through which themixture of host fluid/second fluid or particulate flows into a hollowchamber. The mixture fills up the chamber in the dump diffuser, whichreduces/eliminates flow pulsations and flow non-uniformities that resultfrom pumps, hosing and horizontal inlet flow where gravity effectsdominate. The mixture then flows horizontally out of the dump diffuserand enters the acoustic chamber 107. The dump diffuser brings theheavier mixture into the acoustic chamber (dark arrows) above the bottomof the chamber and below the ultrasonic transducer(s) 106 and the nodalclusters that form in the acoustic standing waves. This minimizes anydisturbances of the clusters set up by inflowing material.

The structure and operation of the dump diffuser is illustrated in FIG.8 and FIG. 9. FIG. 8 is a perspective view of the dump diffuser 530 withthe front plate removed, showing both the interior and the exterior of adump diffuser. FIG. 9 is a perspective view of the front plate of thedump diffuser.

Starting with FIG. 8, the dump diffuser 530 includes a housing 531having an upper end 532, an opposite lower end 534, two side faces 538,a front face 536, and a rear face 539. A hollow chamber 540 is presentwithin the housing 531. The dump diffuser also includes an entrance port542 that receives the mixture and leads into the chamber 540. Theentrance port 542 is present on the upper end and on a side face 538 ofthe housing; two entrance ports are visible here. FIG. 9 is a picture ofthe front plate 546 which is attached to the front face 536 of thehousing. As illustrated here, the diffuser outlet(s) 544 is located onthe lower end 534 and is in the form of two lines of holes, though thesecould also be in the form of a thin slot.

Referring now to both FIG. 2 and FIG. 8, in use, the mixture of hostfluid/second fluid or particulate enters the dump diffuser 530 throughentrance ports 542 and fills up the chamber 540. Pressure then pushesthe mixture uniformly out through diffuser outlets 544. These diffuseroutlets 544 also pass through the sidewall 110 of the device 100, andcan also be considered as the fluid inlet 112 into the interior volume107. The diffuser outlet(s) are placed above the bottom 121 of theacoustic chamber. In embodiments, the diffuser outlets are located abovethe chamber bottom 121 at a height 515 that is between 0% and 100% ofthe height 105 of the acoustic chamber, and more particularly between 5%and 25% of the height of the acoustic chamber. The diffuser outlets 544provide a flow direction parallel to the axial direction of the acousticstanding waves generated by the ultrasonic transducer. The diffuseroutlets are also arranged so that they are in opposing locations, sothat the horizontal velocity of the fluid will decrease to zero in thecenter of the acoustic chamber.

The flow streamlines through the acoustic chamber are desirablysymmetrical, since this minimizes non-uniformities, eddy disturbances,circulation, and disturbance of clusters falling down to concentrateoutlet 116 to be collected. Symmetry also maximizes gravity forces inthe inlet flow distribution and particle collection process. Because itis heavier than the permeate exiting at the top of the device, the(relatively) heavy incoming mixture comes in near the bottom of theacoustic chamber. The symmetrical inlets also assure that the incomingmixture spreads out across the bottom of the chamber due to gravityforces, and provides near uniform velocity profiles from bottom to top.The horizontal velocity of the mixture decreases towards and may equalzero as it approaches the center of the acoustic chamber due to the dualopposing inlet flows. In this example, a uniform velocity contributes toseparation and collection results. The uniform velocity avoids peakvelocities that might prevent the acoustic standing waves fromovercoming particle drag that might impede the clusters from growing andcontinuously leaving the acoustic standing wave via gravity or buoyancyforces.

As the particle clusters drop out, the axial acoustic forces associatedwith the standing wave keep the clusters intact. This effect assuresrapid dropping of the clusters with high terminal velocities, on theorder of 1 cm/sec. This rate is extremely fast compared to the chamberflow velocities, which are on the order of 0.1 cm/sec to 0.3 cm/sec. Theshallow wall angle of the base means the cylindrical particle clusterscan drop a short distance before they exit the acoustic chamber, so thatlittle dispersion of the clusters occurs. Ideally, the system operateswith 3 to 12 trapping lines per square inch of transducer. The symmetry,minimum flow disturbance in the central collection region, and shallowcollector walls provide good collection of the particles.

A second exemplary embodiment of an acoustophoretic device 600 isillustrated in FIGS. 10-12. FIG. 10 is a front cross-sectional view.FIG. 11 is an exterior perspective view of the front of the device. FIG.12 is an exterior perspective view of the rear of the device. In thisdevice, again, the plan cross-sectional area of the acoustic chamber isgreater than the side cross-sectional area of the acoustic chamber.

Starting with FIG. 10, the acoustophoretic device 600 shares manysimilarities with device 100 of FIG. 1. Device 600 has a first side end122 and a second opposite side end 124. A sidewall 110, roof 140 and abase 150 are present to define the interior volume 107. A dump diffuser530 is present on each side end 122, 124, which acts as the fluid inlet112 to the interior volume 107 of the device. Here, the roof 140includes a parabolic interior surface 142 that leads to the fluid outlet114 at the top end 130. Two concentrate outlets 116 are present in thebase 152, with angled walls 152 leading to each outlet at the bottom end132. Five ultrasonic transducers 106 are illustrated, with the rectangleindicating the piezoelectric material used to generate themulti-dimensional acoustic standing wave.

One notable aspect of the device that is more visible in FIG. 10 is therelationship of the placement of the dump diffuser 530/fluid inlet 112to the ultrasonic transducers 106. As seen here, the fluid inlet 112 hasa height 113 that is about 60% of the height 176 of the piezoelectricmaterial. Also, the base 111 of the fluid inlet 112 is located along,i.e. aligned with, the base 177 of the piezoelectric material. Inembodiments, the height of the fluid inlet can be from about 5% to about75% of the height of the piezoelectric material.

Referring now to FIG. 11, the reflector 108 is visible on the front side126 of the device 600. In addition, it can be seen that the fluid outlet114 and the concentrate outlets 116 lead from the top/bottom ends of theinterior volume to the rear side 128 of the device.

Also visible in FIG. 11 is an alternate construction for the dumpdiffuser 530. The dump diffuser of FIG. 8 has two inlet flow ports 542,both located on the side faces 538. In contrast, the dump diffuser ofFIG. 8 has three inlet flow ports 542. Two of the inlet flow ports 542are located on the side faces 538. The third inlet flow port 542 islocated on the rear face 539 on the upper end 532 of the diffuser.

Referring now to FIG. 12, the ultrasonic transducer assembly 170 isseen. Five connectors 171 are visible, one for each of the transducers106 visible in FIG. 10.

Experiments were performed using an acoustophoretic device of FIG. 1.The dimensions of the acoustic chamber were 9 inches by 3 inches by 2inches (length by width by height). The device had six transducersarranged in two rows as illustrated in FIG. 5A. The experiments measuredthe percent reduction/clarification and packed cell mass (PCM) over timeof an incoming water/yeast mixture.

In the graph of FIG. 13, the yeast mixture was 1.5% yeast and was flowedthrough the device at a flow rate of 810 mL/minute. The inlets of thedevice were not part of a dump diffuser (i.e. no front plate asillustrated in FIG. 9 was present). The ultrasonic transducers of thedevice were operated at 60 volts, 80 volts, and 100 volts. The deviceswere operated using acoustically transparent films (ATFs) and alsowithout the use of any such ATFs. As can be seen in FIG. 28, the PCM(lower lines) was measured to be from about 20%-28% at 60 volts, fromabout 28%-35% at 80 volts, and from about 35%-38% at 100 volts, bothwith and without ATFs. The percent reduction/clarification of themixture (upper lines) was about 75%-80% at 60 volts, from about 80%-90%at 80 volts, and from about 85%-90% at 100 volts, both with and withoutATFs, though the devices seemed to have a slightly betterseparation/clarification efficiency without ATFs. Both the PercentReduction and the PCM values suggested that operation at higher voltagesresulted in better separation of water and yeast.

In the graph of FIG. 14, the yeast mixture was 3.0% yeast and was flowedthrough the device at a flow rate of 810 mL/minute. Again, the inlets ofthe device were not part of a dump diffuser. The ultrasonic transducersof the device were operated at 60 volts, 80 volts, and 100 volts. Thedevices were operated using five acoustically transparent films (ATFs).Here, the PCM (lower lines) was measured to be from about 18%-25% at 60volts, from about 25%-29% at 80 volts, and from about 29%-30% at 100volts. The percent reduction/clarification of the mixture (upper lines)was from about 55%-75% at 60 volts, from about 75%-82% at 80 volts, andfrom about 75%-82% at 100 volts.

In the graph of FIG. 15, the yeast mixture was 3.0% yeast and was flowedthrough the device at a flow rate of 810 mL/minute. The inlets of thedevice were part of a dump diffuser with front plates. The darker linesrepresent a dump diffuser wherein the front plate was configured as ahalf plate (i.e. one large slot at the bottom of the front plate), andthe lighter lines represent a dump diffuser where the front plate hadtwo rows of holes. The ultrasonic transducers of the device wereoperated at 80 volts and 100 volts. The devices were operated using fiveATFs. The PCM (lower lines) was measured to be from about 20%-30% at 80volts and from about 30%-35% at 100 volts for the two-rows-holes frontplate. The PCM for the half-plate front plate was about 20%-30% at 80volts and from about 30%-35% at 100 volts. It is noted that the PCM forthe half-plate front plate did not change significantly once the top rowof ultrasonic transducers was turned off. The percentreduction/clarification of the mixture (upper lines) for thetwo-rows-holes front plate was about 68%-80% at 80 volts, and about85%-90% at 100 volts with the top row turned on. The percentreduction/clarification of the mixture for the half-plate front platewas about 68%-80% at 80 volts, from about 85%-90% at 100 volts with thetop row on, and then from about 65%-75% at 100 volts with the top rowturned off.

In the graph of FIG. 16, the yeast mixture was 3.0% yeast and was flowedthrough the device at a flow rate of 810 mL/minute. The inlets of thedevice were part of a dump diffuser, using a half-plate front plate.Only the bottom row of transducers was used, and the ultrasonictransducers of the device were operated at 100 volts. The PCM (lowerline) was measured to be from about 18%-32%, and the percentreduction/clarification of the mixture (upper line) was about 60%-78%.

A third exemplary embodiment of an acoustophoretic device 700 isillustrated in FIGS. 17-22. FIG. 17 is an exterior perspective view.FIG. 18 is a perspective side cross-sectional view of the device. FIG.19 is a side cross-sectional view of the device. FIG. 20 is a frontcross-sectional view of the device. FIG. 21 is a magnified perspectiveside cross-sectional view of the acoustic chamber. FIG. 22 is amagnified perspective side cross-sectional view of the ultrasonictransducer. This particular embodiment is also built in a modularfashion from multiple components.

Starting with FIG. 17, the acoustophoretic device 700 shares somesimilarities with the devices illustrated in FIG. 1 and FIG. 10. Device700 has a first side end 122 and a second opposite side end 124. Asidewall 110, roof 140 and a base 150 are present to define the interiorvolume 107. A dump diffuser 530 is present on each side end 122, 124,which acts as the fluid inlet 112 to the interior volume 107 of thedevice. The dump diffuser here has three inlet flow ports 542, like thatdescribed in the device of FIG. 10. The roof 140 includes a conicalinterior surface 142 that leads to the fluid outlet 114 at the top end130. A concentrate outlet 116 is present in the base 150, with a conicalsurface leading to the concentrate outlet at the bottom end 132. Anultrasonic transducer 106 is present on the rear side, and a reflector108 is present on the front side opposite the transducer.

Referring now to FIG. 18, it can be seen that the fluid outlet 114 andthe concentrate outlets 116 lead from the top/bottom ends of theinterior volume 107 to one of the side ends 124 of the device, i.e. acommon side where a fluid inlet 112 is present. As previously explainedabove, the fluid outlet 112 generally allows for recovery of clarifiedfluid from the interior volume 107. The concentrate outlet 116 generallyallows for recovery or collection of particles, cells.

Referring now to FIG. 19, the interior surface 142 of the roof 140leading to the fluid outlet 114 can be seen, as can the angled walls 152leading to the concentrate outlet 116. The bottom of the acousticchamber is indicated with dotted line 121, and the top of the acousticchamber is indicated with dotted line 119. The interior surface 142 hasan interior angle B measured relative to dotted line 119, with the angleB being in embodiments from about 11° to about 60°, including about 30°to about 45°. Similarly, the angled walls 152 have an angle A measuredrelative to dotted line 121, with the angle A being in embodiments fromabout 11° to about 60°, including about 30° to about 45°. O-rings 180can be disposed between the roof/base and the acoustic chamber so as toprovide a fluid-tight seal therebetween.

Referring now to FIG. 20, it is again seen that the fluid outlet 114 andthe concentrate outlets 116 lead from the top/bottom ends of theinterior volume 107 to one of the side ends 124 of the device, i.e. acommon side where a fluid inlet 112 is present.

The piezoelectric material 178 of the ultrasonic transducer is seen, asis the fluid inlet 112 into the acoustic chamber 107 from the dumpdiffuser 530. The hollow chamber 540 is also seen. The piezoelectricmaterial 178 has a height 176. The fluid inlet 112 also has a height113. The height 113 of the fluid inlet 112 is about 60% of the height176 of the piezoelectric material 178. In embodiments, the height of thefluid inlet can be from about 5% to about 75% of the height of thepiezoelectric material. Again, a bottom edge 111 of the fluid inlet 112is aligned with a bottom edge 177 of the piezoelectric material.

Turning now to FIG. 21, a magnified view of the acoustic chamber 107 ofthe device 700 can be seen, which is sandwiched between the ultrasonictransducer 106 and the reflector 108. A very small gap (e.g., 0.010inches) is present between the fluid inlet 112 and the transducer 106.This gap can be filled with, for example, an O-ring, as depicted in FIG.19. There is also a very short gap (e.g., <0.025 inch) between thebottom edge 177 of the piezoelectric material of the transducer and theangled walls 152 of the base.

FIG. 22 provides a magnified cross-sectional view of the ultrasonictransducer 106. As shown in FIG. 18 and FIG. 19, the ultrasonictransducer 106 is generally located in the sidewall of the device. Asdepicted here, the ultrasonic transducer includes a housing 190. An airgap 194 is present within the housing of the transducer. A connector 191is present and spaced apart from the piezoelectric material 178, whichis in the form of a crystal. A potting material 192, such as epoxy, isused to attach the piezoelectric material 178 to the housing. Anadhesive-backed film 193, made for example from a polyetheretherketone(PEEK), is then attached to the exterior surface of the piezoelectricmaterial 178 and the housing. This film can act as a wear layer. Thewear layer generally has a thickness of a half wavelength or less (e.g.,0.050 inches). Additional features of the ultrasonic transducer(s) usedin the present devices will be explained in greater detail herein.

One specific application for the acoustophoresis devices disclosedherein is in the processing of bioreactor materials. The fluid streamentering these devices is a mixture of a host/primary fluid (e.g. water,cell culture media) and a secondary particulate. The secondaryparticulate can include cells and expressed materials such asbiomolecules (e.g. recombinant proteins or monoclonal antibodies orviruses). The devices can be used to concentrate larger particles, suchas cells, from the mixture, so that there are two different streamsexiting the device. First, a stream of concentrated cells and some fluidcan exit through the concentrate outlet. Second, a stream of clarifiedfluid containing expressed materials such as biomolecules can exitthrough the fluid outlet. Depending on what material is desired to berecovered, either of these two streams exiting the device can berecycled to the bioreactor.

The acoustophoresis devices of the present disclosure, which usethree-dimensional acoustic standing waves, may also be coupled with astandard filtration process upstream or downstream, such as depthfiltration using diatomaceous earth, tangential flow filtration (TFF),or other physical filtration processes, as desired.

Desirably, flow rates through the devices of the present disclosure canbe a minimum of 1 mL/min, or a minimum of about 800 mL/min, anddesirably even higher flow rates can be achieved. In alternate units,these flow rates may be about 0.005 mL/min per cm² of cross-sectionalarea of the acoustic chamber, or about 4.5 mL/min/cm². This is true forbatch reactors, fed-batch bioreactors and perfusion bioreactors.

It may be helpful to provide an explanation now of how multi-dimensionalacoustic standing waves (particularly three-dimensional acousticstanding waves) are generated. The multi-dimensional acoustic standingwave needed for particle collection is obtained by driving an ultrasonictransducer at a frequency that both generates the acoustic standing waveand excites a fundamental 3D vibration mode of the transducer crystal.Perturbation of the piezoelectric crystal in an ultrasonic transducer ina multimode fashion allows for generation of a multidimensional acousticstanding wave. A piezoelectric crystal can be specifically designed todeform in a multimode fashion at designed frequencies, allowing forgeneration of a multi-dimensional acoustic standing wave. Themulti-dimensional acoustic standing wave may be generated by distinctmodes of the piezoelectric crystal such as a 3×3 mode that wouldgenerate multidimensional acoustic standing waves. A multitude ofmultidimensional acoustic standing waves may also be generated byallowing the piezoelectric crystal to vibrate through many differentmode shapes. Thus, the crystal would excite multiple modes such as a 0×0mode (i.e. a piston mode) to a 1×1, 2×2, 1×3, 3×1, 3×3, and other higherorder modes and then cycle back through the lower modes of the crystal(not necessarily in straight order). This switching or dithering of thecrystal between modes allows for various multidimensional wave shapes,along with a single piston mode shape to be generated over a designatedtime.

Some further explanation of the ultrasonic transducers used in thedevices, systems, and methods of the present disclosure may be helpfulas well. In this regard, the transducers use a piezoelectric crystal,usually made of PZT-8 (lead zirconate titanate). Such crystals may havea 1 inch diameter and a nominal 2 MHz resonance frequency, and may alsobe of a larger size. Each ultrasonic transducer module can have only onecrystal, or can have multiple crystals that each act as a separateultrasonic transducer and are either controlled by one or multipleamplifiers. The crystals can be square, rectangular, irregular polygon,or generally of any arbitrary shape. The transducer(s) is/are used tocreate a pressure field that generates forces of the same order ofmagnitude both orthogonal to the standing wave direction (lateral) andin the standing wave direction (axial).

FIG. 23 is a cross-sectional diagram of a conventional ultrasonictransducer. This transducer has a wear plate 50 at a bottom end, epoxylayer 52, ceramic crystal 54 (made of, e.g. PZT), an epoxy layer 56, anda backing layer 58. On either side of the ceramic crystal, there is anelectrode: a positive electrode 61 and a negative electrode 63. Theepoxy layer 56 attaches backing layer 58 to the crystal 54. The entireassembly is contained in a housing 60 which may be made out of, forexample, aluminum. An electrical adapter 62 provides connection forwires to pass through the housing and connect to leads (not shown) whichattach to the crystal 54. Typically, backing layers are designed to adddamping and to create a broadband transducer with uniform displacementacross a wide range of frequency and are designed to suppress excitationat particular vibrational eigen-modes. Wear plates are usually designedas impedance transformers to better match the characteristic impedanceof the medium into which the transducer radiates.

FIG. 24 is a cross-sectional view of an ultrasonic transducer 81 of thepresent disclosure. Transducer 81 is shaped as a disc or a plate, andhas an aluminum housing 82. The piezoelectric crystal is a mass ofperovskite ceramic crystals, each consisting of a small, tetravalentmetal ion, usually titanium or zirconium, in a lattice of larger,divalent metal ions, usually lead or barium, and O₂ ⁻ ions. As anexample, a PZT (lead zirconate titanate) crystal 86 defines the bottomend of the transducer, and is exposed from the exterior of the housing.The crystal is supported on its perimeter by a small elastic layer 98,e.g. silicone or similar material, located between the crystal and thehousing. Put another way, no wear layer is present. In particularembodiments, the crystal is an irregular polygon, and in furtherembodiments is an asymmetrical irregular polygon.

Screws 88 attach an aluminum top plate 82 a of the housing to the body82 b of the housing via threads. The top plate includes a connector 84for powering the transducer. The top surface of the PZT crystal 86 isconnected to a positive electrode 90 and a negative electrode 92, whichare separated by an insulating material 94. The electrodes can be madefrom any conductive material, such as silver or nickel. Electrical poweris provided to the PZT crystal 86 through the electrodes on the crystal.Note that the crystal 86 has no backing layer or epoxy layer. Putanother way, there is an air gap 87 in the transducer between aluminumtop plate 82 a and the crystal 86 (i.e. the air gap is completelyempty). A minimal backing 58 and/or wear plate 50 may be provided insome embodiments, as seen in FIG. 5.

The transducer design can affect performance of the system. A typicaltransducer is a layered structure with the ceramic crystal bonded to abacking layer and a wear plate. Because the transducer is loaded withthe high mechanical impedance presented by the standing wave, thetraditional design guidelines for wear plates, e.g., half wavelengththickness for standing wave applications or quarter wavelength thicknessfor radiation applications, and manufacturing methods may not beappropriate. Rather, in one embodiment of the present disclosure thetransducers, there is no wear plate or backing, allowing the crystal tovibrate in one of its eigenmodes (i.e. near eigenfrequency) with a highQ-factor. The vibrating ceramic crystal/disk is directly exposed to thefluid flowing through the flow chamber.

Removing the backing (e.g. making the crystal air backed) also permitsthe ceramic crystal to vibrate at higher order modes of vibration withlittle damping (e.g. higher order modal displacement). In a transducercomprising a crystal with a backing, the crystal vibrates with a moreuniform displacement, like a piston. Removing the backing allows thecrystal to vibrate in a non-uniform displacement mode. The higher orderthe mode shape of the crystal, the more nodal lines the crystal mayhave. The higher order modal displacement of the crystal creates moretrapping lines, although the correlation of trapping line to node is notnecessarily one to one, and driving the crystal at a higher frequencywill not necessarily produce more trapping lines.

In some embodiments, the crystal may have a backing that minimallyaffects the Q-factor of the crystal (e.g. less than 5%). The backing maybe made of a substantially acoustically transparent material such asbalsa wood, foam, or cork which allows the crystal to vibrate in ahigher order mode shape and maintains a high Q-factor while stillproviding some mechanical support for the crystal. The backing layer maybe a solid, or may be a lattice having holes through the layer, suchthat the lattice follows the nodes of the vibrating crystal in aparticular higher order vibration mode, providing support at nodelocations while allowing the rest of the crystal to vibrate freely. Thegoal of the lattice work or acoustically transparent material is toprovide support without lowering the Q-factor of the crystal orinterfering with the excitation of a particular mode shape.

Placing the crystal in direct contact with the fluid also contributes tothe high Q-factor by avoiding the dampening and energy absorptioneffects of the epoxy layer and the wear plate. Other embodiments mayhave wear plates or a wear surface to prevent the PZT, which containslead, contacting the host fluid. This may be desirable in, for example,biological applications such as separating blood. Such applicationsmight use a wear layer such as chrome, electrolytic nickel, orelectroless nickel. Chemical vapor deposition could also be used toapply a layer of poly(p-xylylene) (e.g. Parylene) or other polymers orpolymer films. Organic and biocompatible coatings such as silicone orpolyurethane are also usable as a wear surface.

FIG. 26 is a log-log graph (logarithmic y-axis, logarithmic x-axis) thatshows the scaling of the acoustic radiation force, fluid drag force, andbuoyancy force with particle radius, and provides an explanation for theseparation of particles using acoustic radiation forces. The buoyancyforce is a particle volume dependent force, and is therefore negligiblefor particle sizes on the order of micron, but grows, and becomessignificant for particle sizes on the order of hundreds of microns. Thefluid drag force (Stokes drag force) scales linearly with fluidvelocity, and therefore typically exceeds the buoyancy force for micronsized particles, but is negligible for larger sized particles on theorder of hundreds of microns. The acoustic radiation force scaling isdifferent. When the particle size is small, Gor′kov's equation isaccurate and the acoustic trapping force scales with the volume of theparticle. Eventually, when the particle size grows, the acousticradiation force no longer increases with the cube of the particleradius, and will rapidly vanish at a certain critical particle size. Forfurther increases of particle size, the radiation force increases againin magnitude but with opposite phase (not shown in the graph). Thispattern repeats for increasing particle sizes.

Initially, when a suspension is flowing through the system withprimarily small micron sized particles, it is necessary for the acousticradiation force to balance the combined effect of fluid drag force andbuoyancy force for a particle to be trapped in the standing wave. InFIG. 26, this happens at a particle size labeled as R_(c1). The graphthen indicates that all larger particles will be trapped as well.Therefore, when small particles are trapped in the standing wave,particles coalescence/clumping/aggregation/agglomeration takes place,resulting in continuous growth of effective particle size. As particlescluster, the total drag on the cluster is much lower than the sum of thedrag forces on the individual particles. In essence, as the particlescluster, they shield each other from the fluid flow and reduce theoverall drag of the cluster. As the particle cluster size grows, theacoustic radiation force reflects off the cluster, such that the netacoustic radiation force decreases per unit volume. The acoustic lateralforces on the particles may be different from the drag forces for theclusters to remain stationary and grow in size. For example, theacoustic lateral forces may be larger than the drag forces to permitparticles to be trapped, cluster and grow in size.

Particle size growth continues until the buoyancy force becomesdominant, which is indicated by a second critical particle size, R_(c2).The buoyancy force per unit volume of the cluster remains constant withcluster size, since it is a function of the particle density, clusterconcentration and gravity constant. Therefore, as the cluster sizeincreases, the buoyancy force on the cluster increases faster than theacoustic radiation force. At the size R_(c2), the particles will rise orsink, depending on their relative density with respect to the hostfluid. At this size, acoustic forces are secondary, gravity/buoyancyforces become dominant, and the particles naturally drop out or rise outof the host fluid. Not all particles will drop out, and those remainingparticles and new particles entering the acoustic chamber will continueto move to the three-dimensional nodal locations, repeating the growthand drop-out process. This phenomenon explains the quick drops and risesin the acoustic radiation force beyond size R_(c2). Thus, FIG. 6explains how small particles can be trapped continuously in a standingwave, grow into larger particles or clumps, and then eventually willrise or settle out because of increased buoyancy force.

The size, shape, and thickness of the transducer determine thetransducer displacement at different frequencies of excitation, which inturn affects particle separation efficiency. Higher order modaldisplacements generate three-dimensional acoustic standing waves withstrong gradients in the acoustic field in all directions, therebycreating equally strong acoustic radiation forces in all directions,leading to multiple trapping lines, where the number of trapping linescorrelate with the particular mode shape of the transducer.

FIG. 27 shows the measured electrical impedance amplitude of thetransducer as a function of frequency in the vicinity of the 2.2 MHztransducer resonance. The minima in the transducer electrical impedancecorrespond to acoustic resonances of a water column and representpotential frequencies for operation. Numerical modeling has indicatedthat the transducer displacement profile varies significantly at theseacoustic resonance frequencies, and thereby directly affects theacoustic standing wave and resulting trapping force. Since thetransducer operates near its thickness resonance, the displacements ofthe electrode surfaces are essentially out of phase. The typicaldisplacement of the transducer electrodes is not uniform and variesdepending on frequency of excitation. Higher order transducerdisplacement patterns result in higher trapping forces and multiplestable trapping lines for the captured particles.

To investigate the effect of the transducer displacement profile onacoustic trapping force and particle separation efficiencies, anexperiment was repeated ten times, with all conditions identical exceptfor the excitation frequency. Ten consecutive acoustic resonancefrequencies, indicated by circled numbers 1-9 and letter A on FIG. 27,were used as excitation frequencies. The conditions were experimentduration of 30 min, a 1000 ppm oil concentration of approximately5-micron SAE-30 oil droplets, a flow rate of 500 ml/min, and an appliedpower of 20 W.

As the emulsion passed by the transducer, the trapping lines of oildroplets were observed and characterized. The characterization involvedthe observation and pattern of the number of trapping lines across thefluid channel, as shown in FIG. 28A, for seven of the ten resonancefrequencies identified in FIG. 27.

FIG. 28B shows an isometric view of the system in which the trappingline locations are being determined. FIG. 28C is a view of the system asit appears when looking down the inlet, along arrow 814. FIG. 28D is aview of the system as it appears when looking directly at the transducerface, along arrow 816.

The effect of excitation frequency clearly determines the number oftrapping lines, which vary from a single trapping line at the excitationfrequency of acoustic resonance 5 and 9, to nine trapping lines foracoustic resonance frequency 4. At other excitation frequencies four orfive trapping lines are observed. Different displacement profiles of thetransducer can produce different (more) trapping lines in the standingwaves, with more gradients in displacement profile generally creatinghigher trapping forces and more trapping lines. It is noted thatalthough the different trapping line profiles shown in FIG. 28A wereobtained at the frequencies shown in FIG. 27, these trapping lineprofiles can also be obtained at different frequencies.

FIG. 28A shows the different crystal vibration modes possible by drivingthe crystal to vibrate at different fundamental frequencies ofvibration. The 3D mode of vibration of the crystal is carried by theacoustic standing wave across the fluid in the chamber all the way tothe reflector and back. The resulting multi-dimensional standing wavecan be thought of as containing two components. The first component is aplanar out-of-plane motion component (uniform displacement acrosscrystal surface) of the crystal that generates a standing wave, and thesecond component is a displacement amplitude variation with peaks andvalleys occurring in both lateral directions of the crystal surface.Three-dimensional force gradients are generated by the standing wave.These three-dimensional force gradients result in lateral radiationforces that stop and trap the particles with respect to the flow byovercoming the viscous drag force. In addition, the lateral radiationforces are responsible for creating tightly packed clusters ofparticles. Therefore, particle separation and gravity-driven collectiondepends on generating a multi-dimensional standing wave that canovercome the particle drag force as the mixture flows through theacoustic standing wave. Multiple particle clusters are formed alongtrapping lines in the axial direction of the standing wave, as presentedschematically in FIG. 28A.

The present disclosure has been described with reference to exemplaryembodiments. Modifications and alterations may occur to others uponreading and understanding the preceding detailed description. It isintended that the present disclosure be construed as including all suchmodifications and alterations insofar as they come within the scope ofthe appended claims or the equivalents thereof.

The invention claimed is:
 1. A method for separating a secondary fluidor particulate from a mixture with a host fluid, comprising: placing themixture in an acoustophoretic device that comprises: an acousticchamber; at least one ultrasonic transducer coupled to the acousticchamber and configured to generate a multi-dimensional acoustic standingwave in the acoustic chamber; a reflector across the acoustic chamberfrom the at least one ultrasonic transducer; and an interior volume ofat least 40 cubic inches; exciting the at least one transducer in ahigher order mode to generate the multi-dimensional acoustic standingwave in the acoustic chamber; and trapping the secondary fluid orparticulate via the acoustic standing wave such that the secondary fluidor particulate agglomerates, aggregates, clusters, or coalesces togetherand separates from the host fluid.
 2. The method of claim 1, furthercomprising flowing the mixture through the acoustophoretic device. 3.The method of claim 2, further comprising flowing the mixture through adump diffuser into the acoustic chamber.
 4. The method of claim 2,further comprising flowing the mixture in a flow direction normal to anaxial direction of the acoustic standing wave generated by the at leastone ultrasonic transducer.
 5. The method of claim 2, further comprisingflowing the mixture into the acoustic chamber via at least two inletsarranged at opposite ends of the acoustic chamber.
 6. The method ofclaim 1, wherein the at least one transducer further comprises aplurality of transducers spanning the length of the acoustic chamber. 7.The acoustophoretic device of claim 6, wherein the plurality oftransducers includes a first row with at least two transducers locatedabove a second row with at least two transducers.
 8. The method of claim1, wherein the multi-dimensional acoustic standing wave includes anaxial force component and a lateral force component which are of thesame order of magnitude.
 9. The method of claim 1, further comprisinggenerating the acoustic standing wave in the acoustic chamber through anacoustically transparent film.
 10. A method for separating a secondaryfluid or particulate from a mixture with a primary fluid, comprising:flowing the mixture of the primary fluid and the secondary fluid orparticulate at a rate of at least 25 mL/min through an acoustophoreticdevice that comprises: an acoustic chamber; at least one ultrasonictransducer coupled to the acoustic chamber and configured to generate amulti-dimensional acoustic standing wave in the acoustic chamber; and areflector across the acoustic chamber from the at least one ultrasonictransducer; exciting the at least one transducer in a higher order modeto generate the multi-dimensional acoustic standing wave in the acousticchamber; and trapping the secondary fluid or particulate via theacoustic standing wave such that the secondary fluid or particulateagglomerates, aggregates, clusters or coalesces together and separatesfrom the primary fluid.
 11. The method according to claim 10, furthercomprising flowing the mixture into the acoustic chamber at a locationbetween about 5% and about 25% of the height of the acoustic chamber.12. The method according to claim 10, further comprising flowing themixture into the acoustic chamber at a rate of at least 800 mL/min. 13.The method according to claim 10, further comprising flowing the mixtureinto the acoustic chamber at a rate in the range of from about 0.005mL/min/cm2 to about 4.5 mL/min/cm2.
 14. The method according to claim10, further comprising flowing the mixture in a flow direction normal toan axial direction of the acoustic standing wave generated by the atleast one ultrasonic transducer.
 15. The method according to claim 10,wherein the wave is multi-dimensional acoustic standing wave thatincludes an axial force component and a lateral force component whichare of the same order of magnitude.
 16. A method for separating asecondary fluid or particulate from a mixture with a primary fluid,comprising: flowing the mixture of the primary fluid and the secondaryfluid or particulate through an acoustophoretic device that comprises:an acoustic chamber; at least one ultrasonic transducer coupled to theacoustic chamber and configured to generate a multi-dimensional acousticstanding wave in the acoustic chamber; and a reflector across theacoustic chamber from the at least one ultrasonic transducer; excitingthe at least one transducer in a higher order mode to generate themulti-dimensional acoustic standing wave in the acoustic chamber throughan acoustically transparent film; and trapping the secondary fluid orparticulate via the acoustic standing wave such that the secondary fluidor particulate agglomerates, aggregates, clusters or coalesces togetherand separates from the primary fluid.